MX2007011396A - Novel soluble epcr protein of non-proteolytic origin and use thereof. - Google Patents

Abstract

The invention relates to a novel soluble form of EPCR originating from an alternative splicing mechanism and, more specifically, to a polypeptide comprising residues 201-256 of sequence SEQ ID NO: 1 or a fragment of said sequence. The invention also relates to a recombinant or isolated EPCR protein comprising said polypeptide, an isolated polynucleotide that codes for said polypeptide, an mRNA encoding a protein that comprises said polypeptide and to the use thereof in the detection of diseases. The invention further relates to an expression vector or a host cell comprising said polynucleotide, an expression system that comprises the expression vector and said cell, a specific isolated antibody for said polypeptide, and a method and a kit for the selective in vitro detection in a biological sample of an EPCR protein comprising sequence SEQ ID NO: 1.

Description

NEW PROTEIN EPCR SOLUBLE OF ORIGIN NO PROTEOLITICQ AND ITS USE BACKGROUND OF THE INVENTION The last protein of the protein C (PC) system described is the endothelial receptor protein C / activated protein C (EPCR) (1, 2). EPCR is expressed in the membrane of endothelial cells and binds with PC and activated protein C (PCA) with high affinity (Kd-30 nM). Its physiological mission is to concentrate the PC on the endothelial surface and present it to the thrombin-thrombomodulin complex, thus favoring an efficient PC activation (3). Studies in nonhuman primates have shown that blockade of EPCR with a monoclonal antibody decreases the physiological production of PCA by 90% and recent studies suggest that genetic defects of EPCR may predispose to venous and arterial thrombosis (4- 7). These facts demonstrate the important role that EPCR can play in the regulation of coagulation and • in thrombosis. But in addition, the EPCR seems to be one of the 'molecules responsible for the powerful anti-inflammatory effect of the PC. The interaction PC / PCA with EPCR could play a crucial role in the modulation of the coagulopathic and inflammatory response that develops in the infection by gram-negative germs (8). Recently, it has been possible to demonstrate that PCA has an anti-inflammatory and anti-apoptotic effect in endothelial cells and that for this effect to be evident, the presence of EPCR is necessary (9,10).

The EPCR is a glycoprotein of approximately 46 kDa. The mature protein is composed of 221 amino acids after the elimination of the signal peptide, which consists of 17 residues (11). It is encoded by a gene located on the long arm of chromosome 20 (20qll.2), formed by four exons interrupted by three introns (12). Exon I encodes the 5 'untranslated region and the signal peptide, exon IV encodes the transmembrane domain, the intracytoplasmic tail composed of three residues and the 3' untranslated region. Exons II and III encode most of the extracellular region of the EPCR, which has homology with the domains a and a.2 of the proteins belonging to the CDl superfamily of the major histocompatibility class I complex. Recently, it has been possible to crystallize and analyze the three-dimensional structure that shows how the EPCR presents a platform formed by folded sheets ß on which two regions with helical conformation are supported, forming a pocket where a phospholipid is housed to stabilize the molecule (13). Thus: ism, by means of alanine scanning, the • main residues involved in the binding with PC / PCA, to which it binds through the GLA domain of it in the presence of calcium and magnesium ions (14). But in addition to the EPCR anchored in the membrane of the endothelial cell, there is a soluble form in plasma (sEPCR) that, at least partially, comes from the digestion of the EPCR by metalloproteinases induced by stimuli such as thrombin (15, 16).

The article (15) J. Clin. Invest. vol. 100, no. 2, July 1997, 411-418, refers to a soluble form of EPCR found in plasma. The EPCR in plasma is said to be a unique species, and the sequencing of the amino terminal fragment of the purified protein resulted in a single EPCR sequence in plasma, and that was coincident with the amino terminal sequence of recombinant sEPCR. It is said that as in the case of other receptors, the origin of said soluble EPCR may be proteolytic cleavage, or an alternative splicing-spindle mechanism, and in particular the genomic structure of bovine EPCR contains an alternative cutting site encoding a protein in which the transmembrane domain is replaced by a sequence encoded by the intron that is located after exon III (17). This document indicates that more studies would be needed to prove the possibility of a form of EPCR derived from alternative cutting. On the other hand, the application O-9900673 refers to the isolation and characterization of sEPCR in human plasma and it is said that the sEPCR can come from proteolysis, in which an extracellular domain of EPCR is released and leaves the rest of the protein bound to the membrane , or of an alternative splicing mechanism of the mRNA that gives rise to the altsEPCR sequence In figure 1 that accompanies the present application, a scheme is shown in which the difference between the soluble EPCR of proteolytic origin is observed (sEPCR ) , he EPCR described in the application WO-9900673 (altsEPCR) obtained By alternative splicing and the new EPCR object of the : present invention (sEPCRvar).

The present application therefore points out that, at least the human EPCR, may undergo an alternative splicing procedure, resulting in soluble truncated EPCR, which includes a single insert present only in the soluble EPCR 'form that comes from said splicing mechanism. alternative splicing (sEPCRvar). Said sEPCRvar can serve as a marker of disease processes in large vessels and cancer. When PCA binds to sEPCR, the former loses its anticoagulant properties but maintains its enzymatic capacity, so it is thought that sEPCR alters the , specificity of the PCA, possibly directing it towards a substrate not known at the moment, which could be in connection with the anti-inflammatory properties attributed to the PCA (18). The sEPCR competes with the EPCR present in the membrane for protein C and thus inhibits the generation of PCA. It has also been described that this sEPCR is capable of interacting with activated neutrophils through proteinase-3 (PR3) (19), a proteinase present in the granules of neutrophils and Mac-1 (CDllb / CD18, an integrin of inducible expression, present on the surface of neutrophils and monocytes). PR3 is .probably involved in mechanisms of tissue destruction acting on membrane-bound TNF-alpha and IL-1beta precursors; Mac-1 is involved in cellular signaling processes and intercellular adhesion phenomena, interacting with adhesion molecules such as CAM-1 endothelial, so it plays a role in the recruitment of neutrophils during the processes inflammatory Presumably, the binding of sEPCR to PR3 and Mac-1 would reduce the activity of these.

Starting from human genomic DNA (cDNA) the inventors have been able to clone and express a new protein. The analysis and comparison of its sequence with known ones, as well as its functional characterization, have shown that this protein is an unknown form of the soluble EPCR protein generated by alternative splicing processes. On the other hand, said protein has as a differential feature a polypeptide region encoded by the region originally considered as 3 'untranslated region of the EPCR gene, which is not present in the EPCR forms known up to now.

DESCRIPTION OF THE INVENTION The present invention relates firstly to a new soluble form of EPCR, which we call variant soluble EPCR (sEPCRvar), more specifically to a polypeptide from a protein C endothelial receptor, which comprises residues 201- 256 of the sequence SEQ ID NO: 1, or a fragment of said sequence of residues. In a particular embodiment said fragment possesses at least 9 amino acids. According to particular embodiments of the invention said fragment is selected from a fragment comprising residues 213-227, 229-242, 212-226, 227-241, 242-256 of SEQ ID NO: 1. In a particular embodiment said fragment possesses immunogenic properties and is particularly useful in procedures for producing specific antibodies. The present invention also relates to an isolated or recombinant protein comprising a polypeptide constituted by residues 201-256 of the sequence SEQ ID NO: 1. Said protein is an EPCR protein. According to a preferred embodiment of the invention, said isolated or recombinant EPCR protein comprises the sequence SEQ ID NO: 1. A further object of the present invention is a recombinant protein comprising a polypeptide, constituted by residues 201-256 of the sequence SEQ ID NO: 1, or a fragment of said sequence of residues. A further object of the present invention is a A polynucleotide comprising a sequence encoding a polypeptide consisting of residues 201-256 of the sequence SEQ ID NO: 1, or a fragment of said sequence of residues. In a particular embodiment said , polynucleotide comes from a cDNA sequence .responding to SEQ ID NO: 2. This cDNA refers to retrotranscribed DNA from mRNA.

In a particular embodiment of the invention the The polynucleotide of the invention can be contained in a 'DNA construct. Thus, a further object of the invention is a DNA construct that encodes a protein or 'polypeptide comprising residues 201-256 of the sequence SEQ ID NO: 1, or a fragment of said sequence of .waste. Preferably said fragment has at least 9 amino acids. In a particular embodiment of said construct, this comprises a polynucleotide derived from a cDNA sequence corresponding to SEQ ID NO: 2. Said DNA construct may incorporate a control sequence operably linked to said protein or polypeptide. When referring to "operably linked" nucleic acids and polynucleotides it means that a nucleotide region is placed in functional relationship with another nucleotide sequence. The "control sequences" are expression signals, specifically recognized by a specific host cell, that regulate functions such as transcription and translation of a specific coding sequence. The promoters control sequences, 'expression enhancers or enhancers, transcription terminators, ribosome binding sites, etc. The splicing of the desired sequences to form the DNA construct can be performed by splicing at appropriate restriction sites. If these binding sites do not exist it is possible to generate them by conventional genetic engineering procedures using synthetic oligonucleotides as adapters or spacers. The polynucleotide or DNA construct of the invention can be obtained by conventional genetic engineering methods, often collected in laboratory manuals. The polynucleotide or construct of the invention can be inserted into a suitable expression vector. Thus, a further object of the present invention is an expression vector comprising said polynucleotide or DNA construct. The choice of the expression vector in the different Embodiments of the invention will depend on the host cell into which said expression vector is to be introduced. By way of example, the vector into which the polynucleotide or DNA construct of the invention is inserted may be a plasmid or a virus, which once introduced into the host cell may or may not be integrated into the cellular genome. This expression vector can also be obtained by conventional methods. A further object of the invention is a transformed host cell comprising a polynucleotide or DNA construct encoding a polypeptide comprising residues 201-256 of the sequence SEQ ID NO: 1, or a fragment of said sequence of residues with minus 9 amino acids According to the invention, the transformed host cell can be a prokaryotic, eg, Escherichia coli, or a eukaryotic cell, e.g. a yeast (among others Pi chia Pastoris and Saccharomyces cerevisiae), an insect cell, or a mammalian cell line. In a particular embodiment of the invention a polynucleotide of the invention with SEQ ID NO: 2 is introduced into Pi chia pastoris to produce a recombinant sEPCR protein of SEQ ID N0: 1, corresponding to the new form of sEPCR generated by alternative splicing described in the present invention (sEPCRvar). The methods used to generate the DNA constructs, The expression vectors and transformed host cells necessary to produce the recombinant protein are described in detail below, including the procedure for their purification. In a particular embodiment of the invention the vector of expression that includes the polynucleotide or DNA construct of the invention is designed for use in compositions and transfer procedures or gene therapy in vivo. In a more particular embodiment this expression vector is a viral vector. Viral vectors suitable for this purpose are, among others, an adenoviral vector, adenoassociated, retroviral, lentiviral, alphaviral, herpesviral, derived from coronavirus, etc. A further object of the present invention is an expression system comprising an expression vector comprising a polynucleotide encoding a polypeptide comprising residues 201-256 of the sequence SEQ ID NO: 1, or a fragment of said sequence of residues, and a host cell transformed with said polynucleotide or said constuct. Preferably said fragment comprises at least 9 amino acids. A further object of the invention is a method for producing a protein or polypeptide comprising residues 201-256 of SEQ ID NO: 1 or a fragment of said sequence of residues, preferably with at least 9 amino acids, said method characterized in that it comprises culturing a host cell comprising a polynucleotide or DNA construct of the invention under conditions that allow the expression of said protein, polypeptide or fragment. The conditions to optimize the culture will depend on the host cell used. If desired, this method may also include some steps for isolation and purification of the expressed protein or polypeptide.

Alternatively, the protein or polypeptide of the invention can be obtained by other conventional methods, for example by chemical synthesis techniques on solid phase; purify by high performance liquid chromatography (HPLC); and, if desired, they can also be analyzed by conventional techniques, for example, by sequencing and mass spectrometry, amino acid analysis, nuclear magnetic resonance, etc. A further object of the present invention is an mRNA that codes for a protein comprising a sequence SEQ ID NO: 1, or fragment of said sequence of residues. A further object of the present invention is an isolated antibody specific for a polypeptide comprising residues 201-256 of SEQ ID NO: 1, or a fragment of said sequence of residues possessing immunogenic properties. In a particular embodiment said fragment possesses at least 9 amino acids. In a particular embodiment said antibody is specific for a polypeptide or fragment whose sequence is selected from residues 201-256, 213-227, 229-242, 212-226, 227-241, 242-256, all of which refer to the SEQ ID NO: 1. In another particular embodiment said antibody is specific for SEQ ID NO: 1 and does not recognize the 1-200 region when it is not bound to region 201-256. According to a preferred embodiment said antibody is a monoclonal antibody. A further object of the present invention is a method for selective detection in vi tro in a sample biological, of an EPCR protein comprising residues 201-256 of the sequence SEQ ID NO: 1, for example the new EPCR protein of SEQ ID NO: 1, or a fragment of said sequence of residues, comprising: - obtaining a biological sample, - analyzing the amount of EPCR protein comprising residues 201-256 of the sequence SEQ ID NO: 1, or a fragment of said residue sequence. Said biological sample can be any sample that includes biological material. In a preferred embodiment said biological sample is a sample of urine, plasma, serum, tissue or interstitial fluid. Said method can be any known method, among others for example mass spectrometry, immunoassays, chemical assays, liquid chromatography and various direct and indirect photometric methods. For example, some analytical methods can be applied to quantify the marker by mass spectrometry. Generally, the label is isolated from the biological sample, for example by liquid chromatography or two-dimensional gel electrophoresis. The marker is quantified by mass spectrometry, for example tandem mass spectrometry associated with liquid chromatography (LC-MS), MALDI-TOF-MS ("matrix-assisted laser desorption / ionization mass spectrometry time-of-flight MS"), etc. The quantification of the target marker can be performed by comparison with standards of the purified marker in known quantities, or by comparison with the amounts of said marker present in the same type of biological samples obtained. of healthy controls. In another embodiment of the invention the method can be an immunoassay. Generally, one or more label ligands are used in the immunoassay. As used in this invention, a "ligand" is any compound or molecule capable of specifically binding to the target marker. These ligands can be used separately or in combination (for example, antibodies in combination with aptamers can be used). In one embodiment of the invention the immunoassay could be a homogeneous assay, a heterogeneous assay, an enzyme-immunoassay (EIA, ELISA), a competitive assay, an immunometric lensay (sandwich), a turbidimetric assay, a nephelometric assay or the like. Similarly, the immunoassay could be performed manually or by means of an automatic analyzer. In the method of the invention said amount of protein is associated with a disease selected from an inflammatory disease associated with vascular damage, inflammation, a disease associated with abnormal coagulation and cancer. Said disease may also be an autoimmune disease such as lupus, sepsis, shok, pre-eclampsia, diabetes, unstable angina, in transplant monitoring, restenosis, angioplasty, liver or kidney disease. According to the method of the invention, it is also possible to comparethe amount of EPCR protein detected against a calibration standard. A further object of the present invention is a kit test for the detection and quantification of an EPCR protein comprising residues 201-256 of the sequence SEQ ID N0: 1, for example the new EPCR protein of SEQ ID NO: 1, or a fragment of said sequence of residues, preferably of at least 9 amino acids, comprising: a) an antibody specific for a polypeptide comprising residues 201-256 of the sequence SEQ ID NO: 1, or a fragment of said sequence of residues, or an antibody specific for the SEQ protein ID N0: 1 that does not recognize region 1-200 when it is not linked to region 201-256. b) reagents for detecting a reaction between the antibody a) and the EPCR protein comprising residues 201-256 of the sequence SEQ ID NO: 1, or a fragment of said residues, present in a biological sample. Preferably the kit also comprises standards for correlating the amount of reaction produced with normal and abnormal levels of EPCR protein comprising residues 201-256 of the sequence SEQ ID NO: 1. A further object of the present invention is the use of a polynucleotide encoding a polypeptide comprising residues 201-256 of the sequence SEQ ID NO: 1, or a fragment of said residue sequence, for the detection and quantification of an mRNA corresponding to an EPCR protein comprising residues 201- 256 of the sequence SEQ ID NO: 1, for example the new EPCR protein of SEQ ID NO: 1, or a fragment of said sequence of residues, preferably said fragment is at least 9 amino acids. This EPCR protein is present in a amount associated with a disease selected from an inflammatory disease associated with vascular damage, inflammation, cancer and a disease associated with abnormal coagulation. In addition, the new soluble form of EPCR of non-proteolytic origin can be used to develop analytical methods that are selective to detect only and exclusively the presence of this form of soluble EPCR, (sEPCRvar), with the advantages that it can entail in the detection of specific diseases, without these methods overlapping with those in which other forms of soluble EPCR or membrane EPCR are detected.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: It shows the different types of EPCR, depending on the way in which the RNA is processed to generate the mature messenger RNA (mRNA) 3 alternative forms of mRNA can be generated, which in turn will produce 3 different forms of the protein: 1. An mRNA with the 4 exons that will be translated as membrane EPCR. 2. An mRNA that includes only exons Ex I, Ex II, and Ex III followed by intron three and the entire sequence subsequent to this intron, and will be translated as a soluble form of EPCR, described by Esmon in O-9900673 (altsEPCR) . 3. By the new form of: alternative splicing described in the invention, an mRNA will be generated that includes exons Ex I, Ex II and Ex III but in the processing the Ex IV exon is lost, with which it will not be transcribed nor the transmembrane domain or the cytoplasmic tail, and instead appears below of exon III a sequence of what would be a cryptic exon located in the 3 'untranslated region, in this case the mRNA will be translated as the new soluble EPCR form of the invention (sEPCRvar). Soluble EPCR can also be generated by proteolytic activity, possibly from a metalloprotease (not yet characterized) that cuts the EPCR at the membrane level, so that a new soluble form of EPCR is generated (sEPCR), which consists of the receptor fraction, extracellular, and lacks the transmembrane domain and the intracytoplasmic tail Figure 2. Banding pattern obtained after amplification of the HUVEC cDNA with EPCR gene specific primers: in addition to the band corresponding to the cDNA of the wild EPCR (1221 bp, arrow 1) a new amplified fragment corresponding to the cDNA is observed of the new variant isoform generated by alternative splicing (arrow 2).

Figure 3. COS-7 cells transfected with wild EPCR (panel A) or with the isoform (panel B), both fused with the green fluorescent protein. Panel A shows how the wild-type protein tends to accumulate in the cell membrane (arrows), whereas with isoform 3 this localization pattern is not seen (panel B).

Figure 4. Illustrates the expression of sEPCRvar in Pichia pastoris. The sEPCRvar was purified from the supernatant of stably transformed P. pastori cells, as indicated in the description. Panel A: Shows different proteins separated by SDS-PAGE and detected using the GELCODE Blue dye; Street St: molecular weight marker, the molecular weight of each band expressed in KDa is indicated; Lane 1: recombinant sEPCR. A diffuse band is observed (due to the intense glycosylation of the protein) of approximately 42 Kda; Street 2: sEPCRvar. A diffuse band (due to the intense glycosylation of the protein) of approximately 47 KDa is observed. Panel B: Proteins detected by Western blot with the anti-EPCR monoclonal antibody (RCR-2); Street St: Molecular weight marker, the molecular weight of each band expressed in KDa is indicated; Street 1: white; Street 2: sEPCRvar. A diffuse band of approximately 47 KDa (ranging between 35 and 60 KDa, due to the variable glycosylation of the protein) is observed.

Figure 5. Amplification of the EPCR gene from cDNA of different tissues and tumors. Panel A) 1: weight marker Molecular, 2: heart, 3: liver, 4: kidney, 5: pancreas, 6: lung, 7: placenta. Panel B) 1: molecular weight marker, 2: skeletal muscle, 3: brain, 4: thymus, 5: small intestine, 6: spleen. Panel C) 1: Marker, 2: prostate, 3: testicle, 4: ovary 5: colon. Panel D) are samples of tumor lines of lung tumors 2: H 446, 3: H 510, 4: H1264, 5: H 549, 6: H 441, 7: HTB 51 8: H 676, 9: H 727, 10: .H 720, 11: H 385, 12: H 1299. The street corresponding to the molecular weight marker is indicated in the figure with the acronym St (standard). The lower band of the marker corresponds to a size of 100 bp; each new band represents a size increased by 100 bp with respect to its lower adjacent band (lOOpb, 200bp, 300bp, ...).

MODE FOR CARRYING OUT THE INVENTION AMPLIFICATION OF EPCR cDNA FROM CELL OF CELLS ENDOTHELIALS HUVEC cells were obtained from human umbilical cord vein, cultured according to standard procedures (Jaffe EA, Nachman RL, Becker CG, Minick CR, Culture of human endothelial cells derived from umbilical veins.) Identification by morphologic and immunologic criteria.J Clin Invest. 1973 Nov; 52 (11): 2745-56). and the total RNA was extracted following standard techniques, as described in (Pérez-Ruiz A, Montes R, Velasco F, López-Pedrera C, Antonio Páramo J, Orb J, Hermida J, Rocha E. Regulation by nitric oxide of endotoxin - induced tissue factor and plasminogen activator inhibitor - 1 in endothelial cells, Thromb Haemost, 2002 Dec; 88 (6): 1060-5.) The obtained RNA was retrotranscribed to cDNA by incubation with transcriptase 60 minutes at 37 ° C, followed by five minutes at 65 ° C for the inactivation of the enzyme. To this end, 120 Units (U) of M-MuLV reverse transcriptase (Gibco BRL) were added to 1 μg of RNA in a final volume of 10 μL, containing 4 μL of the transcriptase buffer solution.

Inverse, 2 mmol / L of deoxynucleotides (dNTPs, Invitrogen), 0.3 μg / mL of random hexamers (Gibco BRL), 0.5 mmol / L of dithiothreitol (DTT, Gibco BRL) and 35 U of inhibitor of Rnasas (human placenta RNAsin 48,000 U / mL, guard RNA, GE Healthcare Bio-Science). The expression of the EPCR gene was analyzed by the polymerase chain reaction (PCR) technique. The primers (Genset, France) used were a 5 'primer corresponding to the sequence SEQ ID NO: 3 and a 3' primer corresponding to the sequence SEQ ID NO. 4. The PCR was carried out in a volume of 25 μL. Aliquots of the cDNA previously obtained by retrotranscription of 1 μg of RNA were used, to which was added 1 U of taq DNA polymerase (Roche), 2.5 μL of reaction buffer solution (Roche), 0.5 μL of each primer (10 μmol / L solution), 0.25 μL deoxynucleotides (dNTPs, GE Healthcare Bio-Science) and 0.25 μl of BSA Img / mL. The reaction mixture is adjusted to 25 microL with water. The amplification cycle consists of a phase of 30 seconds (s) at 94 ° C of denaturation, and another phase of hybridization (annealing) and extension of 3.5 minutes at 72 ° C. The cycles are repeated 5 times after which 32 cycles are performed with the same denaturation phase and a hybridization phase (annealing) and 3 minutes extension at 68 ° C. The amplification is terminated by maintaining the mixture for 5 minutes at 68 ° C as the final extension period. The segment amplified by PCR has a length of 1221 base pairs (bp). When the EPCR gene is amplified under these conditions an additional band appears at a height of approximately 850 bp (Fig. 2).

SEQUENCING OF ADDITIONAL BAND OF AMPLIFIED EPCR The band 2 was reamplified using the same amplification conditions but starting as a template of the 850 bp band of figure 2. The amplification product was purified after agarose gel electrophoresis by means of the kit commercial QIAquick Gel Extraction kit following the manufacturer's instructions. The product thus purified was used as a template for the sequence reaction (ABl Prism® BigDye ™ Terminator Cycle Sequencing Ready Reaction Kit). Two sequence reactions were performed one in the 5 '-3' direction and the other in the opposite direction using the primers corresponding to the sequences SEQ ID NO: 5 and SEQ ID NO. 6 The result of the reaction was analyzed in the automatic sequencer ABl Prism 377 DNA Sequencers. The conclusion is that the new band corresponds to a fragment defined by the sequence SEQ ID NO: 7.

The central part of this fragment whose sequence is SEQ ID NO: 2, corresponds to the cDNA of a new EPCR lacking exon four and part of the 3 'untranslated region. The origin of this cDNA is probably an alternative splicing phenomenon of the EPCR RNA.

This new species of cDNA encodes a polypeptide whose amino acid sequence is SEQ ID NO: l. The residues 1-17 of said sequence correspond to the signal peptide; residues 18-200 correspond to the common extracellular domain of EPCR; residues 201-256 correspond to the new differential domain of this new form of EPCR and replace the transmembrane domain of the EPCR. This new domain does not meet the criteria to be a transmembrane domain.

EXPRESSION OF EPCR AND SEPARATE FUSED WITH GREEN FLUORESCENT PROTEIN IN MAMMALIAN CELLS. To study the subcellular localization of the EPCR 'isoform, its cDNA was cloned into the pcDNA3 vector. l / CT-GFP-TOPO to express it as a protein fused with the green fluorescent protein. The vector thus prepared was used to transiently transform mammalian COS-7 cells in culture using Lipofectamine 2000 (Invitrogen); as a control, the same vector in which the wild-type EPCR cDNA fused to the green fluorescent protein was cloned was used. The subcellular localization of the EPCR .silvestre and the isoform was detected by fluorescence microscopy. As shown in Figure 3, wild-type EPCR is located mainly on the cell membrane, where green fluorescence is observed; however, the sEPCRvar isoform, according to the hypothesis of the inventors, is not localized in the cell membrane.

CLONING, EXPRESSION, PURIFICATION AND CHARACTERIZATION OF THE EPCR ISOFORM Cloning and expression To express the identified variant EPCR (sEPCRvar), the sequence of sEPCRvar without its signal peptide (residues 17-256) was amplified by the polymerase chain reaction (PCR) with the primers 5 '-agcttggcatatcgattagccaagacgcctcagatg-3 ', and 5' -agctatcgtagcggccgcctaccctattatatcagc-3 'that added a restriction site Clal and another Notl at the 5' and 3 'ends respectively, using cDNA from endothelial cells as a template. These modifications allowed to link the sequence of sEPCRvar to the Clal and Notl sites of plasmid pPICZaC (Invitrogen) following the secretion signal of Saccharomyces cerevisiae factor that allows the efficient secretion of many proteins to the extracellular medium from the interior of yeast. Due to the cloning process, a serine residue and an isoleucine residue were added at the amino end of the sEPCRvar. By direct sequencing it was found that the sequence of the insert and the vector was correct. With the expression vector previously prepared and after linearization of it with the restriction enzyme Pmel, Pichia pastoris cells were transformed by a chemical method (Easy Comp, Invitrogen), resulting in the integration, by homologous recombination, of the coding sequence of sEPCRvar into the endogenous methanol response promoter. The product of the transformation was cultured in the presence of zeocin to select those colonies of P. pastoris transformed with the vector containing the coding sequence of the sEPCRvar which in turn contains the gene of the resistance to zeocin. Briefly, the transformed yeasts were cultured in 4 ml of BMY medium [1% (w / v) yeast extract, 2% (w / v) peptone, 100 mM potassium phosphate (pH 6.0), 1, 34% (w / v) of nitrogen source of yeasts with ammonium sulfate, 4x10-5% (w / v) of biotin] supplemented with 1% (v / v) glycerol (BMGY) and incubated at 28-30 ° C for about 18 hours with agitation. The cells were harvested by centrifugation at 2,000 g for 5 minutes at room temperature. The supernatant was discarded and the expression of sEPCRvar with 1% methanol was induced for 18 hours. For this, the cells were resuspended in 3 ml of BMY supplemented with 0.5% (w / v) methanol and incubated for 18 hours at approximately 28-30 ° C with vigorous shaking. After induction, samples from the conditioned medium were loaded on 12% Bis-Tris NuPAGE gels (Invitrogen, Carlsbad, CA) and the sEPCRvar was detected by Western Blot using the monoclonal antibody RCR-2 (Kindly .given by Dr. Kenj i Fukudome, Saga University, Japan) i (figure 4). For large-scale production was selected I the colony that secreted the highest concentration of SEPCRvar. In the colonies selected for their high production capacity of sEPCRvar, the Methanol metabolism (rapid or slow metabolizer) of the colonies, which allowed to establish the optimal expression conditions for the most suitable colony. Once optimized the conditions of cultivation and induction with methanol, the scale was increased to produce large amounts of sEPCRvar.

Purification After concentrating and dialyzing against 20 mM Tris-HCl (pH 7.6) without NaCl, the supernatant of the P pastoris culture, two successive purification steps will be carried out: ion exchange chromatography and gel filtration. In the ion exchange purification, a Resource Q column was used (GE Healthcare Bio-Science) and the elution was performed with a gradient of 0.0-300 mM NaCl in a volume equivalent to that of 20 columns. Eluted fractions containing the sEPCRvar were pooled and concentrated by centrifugal ultrafiltration and then applied to a column to Superdex 75-HR10 / 30 (GE Healthcare Bio-Science) to effect gel filtration. The concentration of purified protein was determined using the BCA total protein assay (Pierre, Rockford, IL) and BSA standards. To detect the purified sEPCRvar, the samples were loaded in 12% gels of NuPAGE Bis-Tris (Invitrogen, Carlsbad, CA) and an electrophoresis was performed, under reducing conditions followed by staining with blue Coomassie An electrophoresis gel was subjected to 'electroblotting and sEPCRvar was detected with the monoclonal antibody RCR-2. To estimate the molecular weight of the sEPCRvar, a molecular weight standard included in each electrophoresis gel was used.

BIOCHEMICAL STUDY TO CHARACTERIZE THE ACTIVITY OF THE NEW VARIANT OF EPCR SOLUBLE The affinity of sEPCRvar for the PC Generation of PCA in cultured endothelial cells. The cell line used was EA.hy926, a transformed human endothelial cell line that has retained the ability to express thrombomodulin and EPCR (Stearns-Kurosawa DJ, Kurosawa S, Molíica JS, Ferrell GL, Esmon CT, The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex, Proc. Natl. Acad. Sci. USA 1996; 93: 10212-6). 5x10"cells were incubated per well of a 96-well plate with 0.02 U / ml thrombin (0.17 nM) (ERL, Swansea, UK) and increasing concentrations of PC (Baxter, Deerfield, IL, USA) ranging from 50 to 1,000 nM in 20 mM Tris buffer, pH 7.4, supplemented with 150 mM NaCl, 5 mM CaCl 2, 0.6 mM MgCl 2, 1% BSA, 0.001% Tween-20 and 0.02% NaN3 After 45 minutes at room temperature lepirudin (Schering AG, Berlin, Germany) was added at a final concentration of 0, 2 μl, to inhibit thrombin and 3-4 minutes later the chromogenic substrate S-2366 (Chromogenix, Milan, Italy) was added to a final concentration of 0.4 mM in order to monitor its proteolysis by PCA. The increase in absorbance at 405 nm was recorded kinetically in a microplate reader (iEMS REader, Labsystems, Finland). The adjustment of the curve data to the Michaelis-Menten equation was performed using the Enzfitter program (Biosoft, Cambridge, UK) which calculated the Km value of the PC activation under those conditions. To study the inhibitory effect of sEPCRvar, 2 μmol / L was added simultaneously with thrombin and PC.

Thus, the effect of sEPCRvar on the activation of the PC could be analyzed and compared with the effect that sEPCR has on PC activation that is a reflection of its affinity for PC. Table 1 shows the biochemical characteristics of the activation of protein C by thrombin on the surface of endothelial cells in culture in the presence or absence of sEPCR and sEPCRvar.

Table 1 The affinity of sEPCR and sEPCRvar for APC Determination of activated partial thromboplastin time. Activated partial thromboplastin time (aPTT) was determined using the Diagnostica Stago Boerhringer Mannheim STA Compact (Roche) kit and the Pathromtin reagent (Dade Behring, USA) using a mixture of five plasmas from healthy subjects. As is well known, the presence of PCA in a mixture of normal plasmas causes the lengthening of the aPTT. Under the experimental conditions used, the aPTT in the absence of PCA was 33.1 ± 0.4 s (mean ± SD), while adding APC to a final concentration of 1 nmol / L, the APTT was prolonged to 43.9 ± 0.4 s. By adding sEPCR the effect of the PCA decreased in such a way that, in the presence of 1 mmol / L of sEPCR, the aPTT was 37.2 + 0.2 s. This effect of sEPCR inhibitor on the anticoagulant effect of PCA is dose dependent. The sEPCR added to the mixture of normal plasmas had no direct effect on the aPTT (33.1 + 0.4 s in the absence versus 33.9 + 0.4 s in the presence of sEPCR). All the experiments were repeated four times. When the same concentration of sEPCR was added instead of sEPCR, the result was superimposable to that obtained with sEPCR, which shows that the sEPCRvar was I joins the PCA, as does the sEPCR. Table 2 shows the effect of sEPCR and sEPCRvar on the anticoagulant function of PCA. Coagulation times (APTT) of a mixture of normal plasmas to which was added .1 nmol / L of PCA and different concentrations of sEPCR. The mean ± SD of four experiments is represented. The aPTT in the absence of PCA was 33.1 ± 0.4 seconds.

Table 2 STUDY OF THE EXPRESSION OF SEPCRvar IN DIFFERENT TISSUES AND IN TUMOR LINES To identify the tissues in which sEPCRvar is expressed, a cDNA library from different human tissues (Multiple tissue cDNA (MTC) panels I and II, Bioscience, USA) was studied by PCR using primers that allow the simultaneous amplification of the cDNA corresponding to EPCR and sEPCRvar. The primers (Genset, France) used were Primer 5 ': 5' -GCAGTATGTGCAGAAACATATTTCCGC-3 ', and Primer 3': 5 '-CATCCCAAGTCTGACACACCTGGAAGT-3'. The PCR was carried out in a volume of 25 μL. Aliquots of the cDNA were used, to which were added 1 U of taq DNA polymerase (Roche), 2.5 μL of reaction buffer solution (Roche), 0.5 μL of each primer (from a 10 μmol / L solution). ), 0.25 μL of deoxynucleotides (dNTPs, GE Healthcare Bio-Science) and 0.25 μl of BSA lmg / mL. The reaction mixture is adjusted to 25 microL with water. The amplification cycle consists of a phase of 30 seconds (s) at 94 CC of denaturation, and another phase of hybridization (annealing) 1 MINUTE at 56 ° C and extension of 1.5 minutes at 72 ° C. The cycles are repeated 35 times. The amplification is finished keeping the mixture for 5 minutes at 72 ° C as the final extension period. The amplified EPCR cDNA segment has a length of 578 bp while that of the sEPCRvar is 189 bp (Fig. 5). The sEPCRvar form is expressed 'abundantly in pancreas, heart, liver, kidney, lung, placenta, thymus, small intestine, spleen and colon. The shape; sEPCRvar is abundantly expressed in some of the tumor lines especially in H 549, H 441 and H 720. Some of the tumor lines, particularly H 446 and H 676, also express another form not yet identified that according to their Electrophoretic mobility has 1000 bp.

ANTIBODY PRODUCTION | Immunization. Male BALB / C mice were immunized from at least 1 month of age with sEPCRvar according to the following pattern: Three subcutaneous or intradermal, or mtraperitoneal immunizations, or combinations of these with an immunogen amount between 10 and 200 μg in Freund's adjuvant or other adjuvant, spaced at least two weeks; Two weeks after the third immunization, two new booster doses were administered, spaced two days and using the same amount of immunogen but this time in saline. Other animals were also immunized with a mixture of the peptides 213-227, 229-242, 212-226, 227-241, 242-256 of SEQ ID NO: 1. These peptides were chosen as being highly hydrophilic fragments in accordance with the Protscale program following the algorithm of Kyte J., Doolittle RF (J. Mol. Biol., 1982; 157: 105-132) Fusion. Two days after the last booster dose, hybridomas will be produced; technique consisting in the fusion of animal splenocytes with myeloma cells (SP2 / 0-Agl4, -P3X63-Ag8.6.5.3, P3 -NS- 1 -Ag4-1, etc.) in the presence of polyethylene glycol 1500 or 4000 This agent makes possible the fusion of the membranes, so that hybrids will be generated between the different cell types: the hybrid between a B lymphocyte of the animal and a myeloma cell will constitute a new cell type, hybridoma, which will produce an antibody and will also be immortal in culture. HAT medium will be added to the cell culture to select these hybridomas and eliminate the rest of the hybrids: the HAT contains aminopterma, an inhibitor of the de novo pathway of guanosine (via De novo). Because the myeloma cells lack functional HPRT enzyme necessary in the salvage route of nucleic acid synthesis, when they are blocked with aminopterin the only available route, via de novo, are unable to synthesize nucleic acids; therefore, after two weeks of culture, the only cells that will survive in the culture will be some splenocyte-myeloma hybrids: the splenocytes will confer the ability of the hybrid to the synthesis of guanosine by the alternative route, and the myeloma cell the ability to be ' cultivated indefinitely in in vi tro culture.

'Detection of hybridomas producing anti-sEPCRvar and anti sEPCR antibodies. The cells in HAT medium will be distributed in at least ten 96-well culture microplates. Between nine and fourteen days after the fusion, the size of the colonies will be sufficient to analyze the presence of antibodies in its supernatant. In order to select the hybridomas that secrete antibodies that interest us, that is, antibodies against the sEPCRvar, supernatant samples will be taken from all the wells of .the five microplates containing clones for an immunoassay: ELISA and Western Blot assays will be carried out. For the ELISA plates will be upholstered with sEPCRvar 0.3 μg / well; After an incubation of 15 hours at 4 ° C and the corresponding blockade with suitable protein, the supernatants of the cultures will be added, the corresponding washes will be made and then a secondary anti-mouse immunoglobulin antibody. In this way, after washing and revealing with peroxidase and o-phenylenediamine wells in which color is detected or an increase in absorbance (492 nm) may contain clones of antibody-secreting hybridomas against sEPCRvar. For the Western Blot a polyacrylamide gel will be run with the recombinant protein and / or a tissue or cell extract that expresses the sEPCRvar protein. It will be transferred to a nitrocellulose or PVDF membrane that will be divided into separate zones by a grid-like apparatus to test the presence of specific antibodies in the supernatants. After blocking and incubation with the specific antibody, it will be washed three times and incubated with a secondary antibody conjugated to the enzyme peroxidase. The development is performed by a substrate that when transformed by the enzyme gives rise to light in the place where the specific antibody has been bound. If the light band corresponds to a protein with the expected molecular weight, the antibody producing cells of the well from which the supernatant comes will grow and freeze in liquid nitrogen. Since sEPCRvar contains the entire sEPCR portion, the reactivity to the extracellular region of recombinant EPCR of hybridomas that are positive for sEPCRvar will be studied. In this way, hybridomas that produce antibodies against the specific region of sEPCRvar can be selected.

Monoclonality of the hybridomas. To ensure that each cell culture that secretes an anti-sEPCRvar antibody is monoclonal, cloning techniques will be applied. limit dilution: isolated cells will be grown in new culture microplates from the original culture or stem cells that tested positive in the first Western Blot or ELISA assay. Once the new colonies emerged from one or more cells have acquired sufficient size, they will be taken again supernatants to submit them to a new ELISA or Western Blot. The colonies that give positivity again will be subjected to limit dilution and the process will be repeated until 100% of the supernatants analyzed after the third limit dilution contain antibodies against the sEPCRvar.

Purification of antibodies. After cultivating the hybridomas under special conditions to obtain a higher yield in antibody concentration (culture bottles with the CELLine system of Integra BioSciences or with the CELline CL-1000 system of Becton Dickinson), the supernatants containing IgGs will be subjected to purification by liquid chromatography using one or more of the following methodologies: immunoaffinity chromatography, affinity chromatography (protein A / Protein G / Protein L, immobilized metal, thioaffinity), cation exchange, hydroxyapatite, hydrophobic interaction, gel filtration, etc. Using an AKTA FPLC team, GE Healthcare Bio-Science).

BIBLIOGRAPHY 1. Bangalore N, Drohan WN, Orthner CL. High affinity binding sites for activated protein C and protein C on cultured human umbilical vein endothelial cells. Independent of protein S and distinct from known ligands. Thromb Haemost 1994; 72: 465-74. 2. Fukudome K, Esmon CT. Identification, cloning, and regulation of a novel endothelial cell protein C / activated protein C receptor. J Biol Chem 1994; 269: 26486-91. 3. Stearns-Kurosawa DJ, Kurosawa S, Mollica JS, Ferrell GL, Esmon CT. The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex. Proc Natl Acad Sci USA 1996; 93: 10212-6. 4. Taylor FB Jr, Peer GT, Lockhart MS, Ferrell G, Esmon CT. Endothelial cell protein C receptor plays an important role in protein C activation in vivo. Blood 2001; 97: 1685-8. 5. Biguzzi E, Merati G, Liaw PC, Bucciarelli P, Oganesyan N, Qu D, Gu JM, Fetiveau R, Esmon CT, Mannucci PM, Faioni EM. TO 23bp insertion in the endothelial protein C receptor (EPCR) gene impairs EPCR function. Thromb Haemost 2001; 86: 945-8. 6. Saposnik B, Reny JL, Gaussem P, Emmerich J, Aiach M, Gandrille S. A haplotype of the EPCR gene is associated with increased plasma levéis of sEPCR and is a candidate risk factor for thrombosis. Blood 2004; 103: 1311-1318. 7. Uitte de Willige S, Van Marión V, Rosendaal FR, Vos HL, Visser MC, Bertina RM. Haplotypes of the EPCR gene, plasma sEPCR levéis and the risk of deep venous thrombosis. J Thromb Haemost. 2004; 2: 1305-10. 8. Taylor FB Jr, Stearns-Kurosawa DJ, Kurosawa S, Ferrell G, Chang AC, Laszik Z, Kosanke S, Peer G, Esmon CT. The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis. Blood 2000; 95: 1680-6. 9. Joyce DE, Gelbert L, Ciaccia A, DeHoff B, Grinnell BW. Gene expression profile of antithrombotic protein defines new mechanisms modulating inflammation and apoptosis. J Biol Chem. 2001; 276: 11199-203. 10. Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science 2002; 296: 1880-2. 11. Fukudome K, Kurosawa S, Stearns-Kurosawa DJ, He X, Rezaie AR, Esmon CT. The endothelial cell protein C receptor. Cell surface expression and direct ligand by the soluble receptor. J Biol Chem 1996; 271: 17491-8. 12. Simmonds RE, Lane DA. Structural and functional implications of the intron / exon organization of the human endothelial cell protein C / activated protein C receptor gene. Comparison with the structure of CDl / major histocompatibility complex a 1 and a 2 domains. Blood 1999; 94: 632-41. 13. Oganesyan V, Oganesyan N, Terzyan S, Qu D, Dauter Z, Esmon NL, Esmon CT. The crystal structure of the endothelial protein C receptor and a bound phospholipid. J Biol Chem '2002; 277: 24851-4. 14. Liaw PC, Mather T, Oganesyan N, Ferrell GL, Esmon CT. Identification of the protein C / activated protein C binding sites on the endothelial cell protein C receptor. Implications for a novel mode of ligand recognition by a major histocompatibility complex class 1-type receptor. J Biol Chem 2001; 276: 8364-70. 15. Kurosawa S, Stearns-Kurosawa DJ, Hidari N, Esmon CT.

Identification of functional endothelial protein C receptor in human plasma. J Clin Invest 1997; 100: 411-8. 16. Xu J, Qu D, Esmon NL, Esmon CT. Metalloproteolytic reagent of endothelial cell protein C receptor. J Biol Chem 2000; 275: 6038-44. 17. Fukudome K, Esmon CT. Molecular cloning and expression of murine and bovine endothelial cell protein C / activated protein C receptor (EPCR). The structural and functional conservation in human, bovine, and murine EPCR. J Biol Chem 1995; 270: 5571-7. 18. Liaw PCY, Neuenschwander PF, Smirnov MD, Esmon CT. Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C. J Biol Chem 2000; 275: 5447-52. 19. Kurosawa S, Esmon CT, Stearns-Kurosawa DJ. The soluble endothelial protein C receptor binds to activated neutrophils: involvement of proteinase-3 and CDllb / CD18. J Immunol 2000; 165: 4697-703.

Claims (22)

1. An EPCR protein, isolated or recombinant Endothelial Protein C Receptor characterized in that it comprises a polypeptide of sequence SEQ ID NO: 12.

2. An isolated or recombinant EPCR protein according to claim 1, characterized in that it comprises the sequence SEQ ID NO: 1.

3. A polypeptide derived from the protein described in claim 2, characterized in that it comprises SEQ ID NO: 12 or a fragment thereof of at least 14 amino acids. ,

4. A polypeptide according to claim 3, characterized in that it is a fragment selected from: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.

5. An isolated or recombinant polynucleotide, characterized in that it encodes an EPCR protein of sequence SEQ ID NO: l.

6. A polynucleotide according to claim 5, characterized in that it is derived from a cDNA sequence corresponding to SEQ ID NO: 2.

7. An expression vector, characterized in that it comprises a polynucleotide defined in one of claims 5 or 6.

8. A host cell, characterized in that it comprises a polynucleotide defined in one of claims 5 or 6. 6, or an expression vector according to claim 7.

9. A host cell according to claim 8, characterized in that the polynucleotide is operably linked to an appropriate control sequence.

10. A host cell according to one of claims 8 or 9, characterized in that said cell is a prokaryotic cell or a eukaryotic cell.

11. A host cell according to claim 10, characterized in that said cell is the bacterium Escherichia coli or the yeast Pi chia pas toris.

12. An expression system characterized in that it comprises an expression vector according to claim 7 and a host cell according to one of claims 8 to 11.

13. A method for producing an EPCR protein of SEQ ID NO: 1, characterized in that it comprises cultivating a host cell containing a polynucleotide of SEQ ID NO: 2 under conditions that allow the expression of said protein.

14. An mRNA characterized in that it encodes a protein comprising the sequence SEQ ID NO: 12, or fragments of said sequence of residues of at least 14 amino acids.

15. An isolated antibody characterized in that it specifically recognizes an EPCR protein according to claim 1 or a polypeptide according to claim 3 which has immunogenic properties. .

16. An isolated antibody according to claim 15, characterized in that it is specific for a polypeptide or fragment of said polypeptide whose sequence has been selected between SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17

17. An antibody according to claim 15, characterized in that it is specific for SEQ ID NO: 1 and because it does not recognize the 1-200 region when it is not bound to region 201-256 (SEQ ID NO: 12). ).

18. An antibody according to one of claims 15 to 17, characterized in that it is a monoclonal antibody.

19. A method for selective detection in vi tro in a biological sample of an EPCR protein comprising the sequence SEQ ID NO: 12, characterized in that it comprises: - obtaining a biological sample, and - analyzing the amount of EPCR protein comprising the SEQ ID NO: 12.

20. A method according to claim 19, characterized in that said amount of protein is associated with a disease selected from an inflammatory disease associated with vascular damage, inflammation, cancer and a disease associated with abnormal coagulation.

21. A method according to one of claims 19 or 20, characterized in that it comprises comparing the amount of EPCR protein detected against a calibration standard.

22. A test kit for the detection and quantification of an EPCR protein comprising the sequence SEQ ID NO: 12, characterized in that it comprises: a) a specific antibody defined in one of claims 15 to 18, and b) reactive to detect a reaction between the antibody a) and the EPCR protein comprising the sequence SEQ ID NO: 12 present in a biological sample.